1. Field of the Invention
The present invention is directed to hybridoma cell lines which produce monoclonal antibodies specific for glycosphingolipids and to the monoclonal antibodies produced by the hybridomas. More particularly, the invention is directed to hybridomas which produce monoclonal antibodies recognizing an abnormal branching structure in a glycosphingolipid. The invention is further directed to a method of chemically synthesizing a glycosphingolipid, and a method for detecting a cancer-associated glycosphingolipid using the monoclonal antibodies of the invention.
2. Description of the Related Art
In 1975, Kohler and Millstein introduced a procedure for the production of monoclonal antibodies using hybrid cells (hybridomas) which allows the production of almost unlimited quantities of antibodies of precise and reproducible specificity. Before that time, conventional antisera which were produced by immunizing animals with tumor cells or other antigens were found to contain a wide variety of different antibodies which differed in their specificity and properties. On the other hand, hybridomas produce a single antibody with uniform characteristics. The Kohler-Millstein procedure entails the fusion of spleen cells from an immunized animal with an immortal myeloma cell line. From the fused cells, known as hybridomas, clones are selected that produce antibody of the desired specificity. Each clone continues to produce only that one antibody. As hybridoma cells can be cultured indefinitely or stored frozen in liquid nitrogen, a constant supply of that particular antibody is assured.
Antibodies are proteins that have the ability to combine with and recognize other molecules, known as antigens. Monoclonal antibodies are no different from other antibodies except that they are very uniform in their properties and recognize only one antigen or a portion of an antigen known as a determinant. Not all of the hybridoma clones which result from fusing neoplastic cells with antibody producing cells are specific for the desired foreign substance or antigen because many of the hybridomas will make antibodies which the innoculated animal has produced to react with other foreign substances. Even antibodies against the subject antigen will differ from clone to clone because antibodies produced by different cells may react with different antigenic determinants of the same molecule. Thus, it is impossible to predict in advance which particular portion of the antigen molecule a particular monoclonal antibody might recognize. Many of the techniques for producing monoclonal antibodies and hybridomas are now routine, as evidenced by the recent book "Monoclonal Hybridoma Antibodies: Techniques and Applications" (edited by John G. Hurrell, 1983), which is hereby incorporated by reference.
The present invention is directed in particular to monoclonal antibodies (and hybridomas producing them), which recognize certain carbohydrate antigens associated with cancer. Carbohydrate antigens have been shown to be important cancer-associated antigens by several researchers. See Hakomori, S. & Kannagi, R., J. Natl. Cancer Inst. 71, 231-251; Feizi, T., Nature 314, 53-54, 1985; Hakomori, S., Cancer Res. 45, 2405-2414, 1985; and Magnani, J. L., Biochem. Soc. Trans. 12, 543-545, 1984, each of which is incorporated herein by reference. Several monoclonal antibodies raised against cancer cells have been shown to recognize abnormal cell surface carbohydrate antigens carried by glycolipids, or, less frequently, by glycoproteins. Some of these antibodies are already applied to measure the presence of antigens in the sera of patients with various cancers. See Koprowski, H. et al., Science 212, 53-55, 1979; Metzger, et al, Cancer Res. 42, 601-608, 1982; Bast, R. et al, N. Eng. J. Med. 309, 883-887, 1983.
More recently, cancer-associated glycolipid antigens extracted and purified from various human tumor cells have been directly utilized for immunization of mice, and several monoclonal antibodies useful for the detection of human cancer have been established. See Hakomori, S. et al, J. Biol. Chem. 259, 4681-4685, 1984; Fukushi, Y. et al, J. Biol. Chem. 259, 10511-10517, 1984; Fukushi, et al, Cancer Res. 45, 3711-3717, 1985; and Kannagi et al., Cancer Res. 46, 2619-2626, 1986.
In spite of the above successes in producing antibodies to glycolipid antigens, some cancer-associated glycolipids are very minor membrane components, and frequently it is not easy to purify them from natural sources in a satisfactory amount for the preparation of monoclonal antibodies. Even though progress has been made in the methodology for purification of glycolipid antigens, the small amounts of these materials has rendered monoclonal antibodies to them impossible to obtain in some instances. In such cases, it would be better to obtain the glycolipid antigen through chemical synthesis, and then to obtain monoclonal antibodies directed to the synthetic antigens. However, the chemical synthesis of these glycolipids is greatly complicated by their stringent structural requirements, such as unusual branching structures, stereochemical configurations, large molecular weights and other complex structural features. Moreover, synthetic schemes are often not feasible because of low yields in required reactions, and the long reaction sequences involved.
Another complicating factor in obtaining monoclonal antibodies specific to cancer-associated glycolipids is that the desired monoclonals must recognize a particular feature of the glycolipid such as an abnormal branching portion. At the same time, the monoclonals must not cross-react with portions of the antigen molecule which are present on the individual segments of the assembled antigen molecule.
In view of the above difficulties, there continues to remain a need for new methods of obtaining monoclonal antibodies to rare antigens, such as cancer-associated glycolipid antigens, and for the monoclonal antibodies themselves.